The authors have declared that no competing interests exist.
Conceived and designed the experiments: JTO SAA IJE. Performed the experiments: REW MPS. Analyzed the data: JTO LDC. Contributed reagents/materials/analysis tools: MJT EAL. Wrote the paper: JTO SAA IJE.
Breast cancers that over-express a lipoxygenase or cyclooxygenase are associated with poor survival possibly because they overproduce metabolites that alter the cancer’s malignant behaviors. However, these metabolites and behaviors have not been identified. We here identify which metabolites among those that stimulate breast cancer cell proliferation in vitro are associated with rapidly proliferating breast cancer.
We used selective ion monitoring-mass spectrometry to quantify in the cancer and normal breast tissue of 27 patients metabolites that stimulate (15-, 12-, 5-hydroxy-, and 5-oxo-eicosatetraenoate, 13-hydroxy-octadecaenoate [HODE]) or inhibit (prostaglandin [PG]E2 and D2) breast cancer cell proliferation. We then related their levels to each cancer’s proliferation rate as defined by its Mib1 score.
13-HODE was the only metabolite strongly, significantly, and positively associated with Mib1 scores. It was similarly associated with aggressive grade and a key component of grade, mitosis, and also trended to be associated with lymph node metastasis. PGE2 and PGD2 trended to be negatively associated with these markers. No other metabolite in cancer and no metabolite in normal tissue had this profile of associations.
Our data fit a model wherein the overproduction of 13-HODE by 15-lipoxygenase-1 shortens breast cancer survival by stimulating its cells to proliferate and possibly metastasize; no other oxygenase-metabolite pathway, including cyclooxygenase-PGE2/D2 pathways, uses this specific mechanism to shorten survival.
The growth rate of cancer is commonly estimated by measuring a cell proliferation-related protein, Ki-67, with Mib1 monoclonal antibody. The Mib1 score provides a particularly strong prognostic marker in human breast cancer
5-LO and 12-LO are over-expressed in the breast cancer of patients who suffer poor survival
Over-expression of CO-2 in breast cancer has been reported to reduce survival
The varying results of the cited studies may reflect an imperfect relation between levels of the oxygenases and their metabolites: an oxygenase’s level does not indicate its activity status, its substrate availability, or the action of other oxygenases which make the same metabolite(s). To clarify the roles of the oxygenases in breast cancer, levels of their metabolites need to be defined and, similar to their parent enzymes, related to factors in the disease that alter survival. Radioimmunoassay studies conducted >20 years ago found that PG-like material in breast cancer was associated with an increase, no change, or a decrease in survival
We measured the metabolites in the malignant and normal breast tissue and the fatty acids (FA) in malignant and normal breast tissue, plasma, and RBC of 27 women undergoing surgery for breast cancer at Wake Forest University Medical Center. The study was approved by the Institutional Review Board of Wake Forest University School of Medicine and IRB-approved, written informed consent was obtained from each subject before surgery. Consenting patients were taken in sequence with no knowledge of their disease status; patients with recurrent disease, inflammatory breast cancer, a malignancy other than adenocarcinoma, in situ disease, neo-adjuvant treatment, or failure to have a Mib1 score were excluded retrospectively. All patients included in the study were diagnosed as having invasive ductal carcinoma and were categorized by the following assessments of their cancer: Mib1 score of ≤20 vs. >20 (13 vs. 14 patients, respectively); grade I and II vs. III (Nottingham score [Bloom-Richardson grading system] of 3–5 vs. 6–9; 10 vs. 17 patients) and grade’s component indices, mitosis (2–3 vs. 1; 19 vs. 8 patients), nuclear pleomorphism (3 vs. 2 & 1 19 vs. 8 patients; only 1 patient had a score of 1 [omitting this patient did not effect the statistical significance of results]), and tubule formation (3 vs. 2, 24 vs. 3 patients)(no patient had a tubule index of 1); Her2 negative vs. positive (histological score of 0 or I vs. II or III; 21 vs. 6 patients); estrogen receptor negative vs. positive (histological score of ≤10 vs. >10; 12 vs. 15 patients); progesterone receptor negative vs. positive (histological score of ≤10 vs. >10; 15 vs. 12 patients); triple negative vs. receptor positive for Her2, estrogen, and/or progesterone receptors (12 vs. 15 patients); and tumor size I vs. II–IV (<2 cm vs. ≥2 cm or spread to chest wall or skin; 13 vs. 14 patients). Patients were also categorized based on self-reported race of Caucasian vs. African American (21 vs. 6 patients; no Hispanic, Oriental, or Native Americans occurred in the patient sequence studied); age >50 or ≤50, (7 vs. 20 patients); lymph node metastasis absent (N0) vs. present (N1–N3; 21 vs. 6 patients); and body mass index (BMI) of ≤30 vs. >30 (20 vs. 7 patients).
Breast tissue was removed by lumpectomy or mastectomy and identified visually as malignant (subject to histological confirmation) or normal. Within 15 minutes of removal for lumpectomy or mastectomy specimens, malignant and normal (taken from sites >0.5–1 and >2 cm, respectfully, from malignant tissue) tissues were sampled, placed on ice and dissected into 3 approximately equal-sized specimens (two for metabolites, one for FA). Specimens designated as malignant and normal from the same patient were immediately weighed (wet weight of 3–110 mg for malignant, 5–90 mg for normal) and within 20 min of dissection, separately processed for content of metabolites and FA.
Precursor | Product | Precursor | Product | ||
Analog | ion−1m/z | ion−1m/z | Analog | ion−1m/z | ion−1m/z |
PGE2 | 351 | 271 | d7-5-oxo-ETE | 324 | 210 |
d4-PGE2 |
355 | 275 | LTB4 | 335 | 194.8 |
PGE3 | 349 | 269 | d4-LTB4 | 339.2 | 196.8 |
PGD2 | 351 | 271 | 15-HETE | 319 | 218.8 |
d4-PGD2 | 355 | 275 | d8-15-HETE | 327 | 225.7 |
5-HETE | 319 | 115 | 12-HETE | 319 | 179 |
d8-5-HETE |
327 | 115.8 | d8-12-HETE | 327 | 184 |
5-HEPE | 317 | 115 | 13-HODE | 295 | 194.7 |
5-oxo-ETE | 317 | 203 | d4-13-HODE | 299 | 197.7 |
Internal standard for PGE2 and PGE3.
Internal standard for 5-HETE and 5-HEPE.
Breast tissue extracts were quantified for metabolites by measuring their precursor and parent ions and correcting for processing losses based on the recovery of their deuterated internal standards by liquid chromatography (LC)-tandem MS running in the multiple reaction-monitoring mode.
Just before analysis, specimens were spiked with an internal FA standard, extracted, ran on gas chromatography, and quantified as described
Deuterated metabolites (Cayman Chemical) and MCF-7 and MDA-MB-231 breast cancer cell lines (American Type Culture Collection (Manassas, VA) were purchased. We prepared 13-HODE and 15-HETE by incubating LA and AA, respectively, with soybean type II LO and 5-oxo-ETE by chemically oxidizing 5-HETE as described
Cell growth was assayed with Cell Titer96 Aqueous One Solution Cell Proliferation Assays (Promega) as described
Cell growth data are given in OD units at 490 nm for cultures processed in the Promega assay. Metabolite levels are reported as pg/mg of wet tissue weight; FA levels are in µg/mg of wet tissue weight or percentage of total recovered FA. Differences between outcomes observed in normal and malignant tissue were assessed using paired t-tests while differences in outcomes between independent groups (e.g., grade I/II vs. III) were assessed using Student t-tests with the Welch correction used if the equality of variances p-value was <0.05. Correlations between parameters are presented as Pearson coefficients. All probability tests were two-sided with their significances being corrected, where indicated, for multiple observations by the false discovery rate method
Since the effects of 13-HODE and 15-HETE on growth of breast cancer cells are unclear, we tested them for this. Both metabolites increased cell number in MDA-MB-231 and MCF-7 cultures at ≥100 pM, achieving responses similar to the known
MCF-7 (upper panels) and MDA-MB-231 (lower panels) cell cultures were challenged with a metabolite for 48 hr and assayed for cell density. One-way ANOVA gave the statistical significances shown between comparisons of cells treated with 0 (culture media) or 100 pM–1 µM of the indicated metabolite. Data are means ±SEM in ODU490 of 3–6 cultures.
We measured the 10 metabolites and 8 deuterated internal standards listed in
Levels of the metabolites are compared by tissue type (panel A), Mib1 score in malignant (panel B) or normal (panel C) tissue; and grade in malignant tissue (panel D). Probability values were defined by paired (panel A) or unpaired (panels B, C, and D) Student t-tests and were corrected for the 7 comparisons made in each panel by the false discovery rate method.
Mib1 scores of >20 and ≤20 classify breast cancer into respectively faster and slower proliferating diseases with corresponding poorer or better survivals. In breast cancer tissue, 13-HODE stood alone in being significantly related to this classification: it was >3.3-fold higher in patients with >20 Mib1 scores (
Our study population had 1 grade I, 9 grade II, and 17 grade III tumors. Increasing grade predicts increasingly more aggressive disease and poorer survival. We compared grade I and II to grade III (omitting grade I did not alter the significance of this comparison). In malignant tissue, 13-HODE levels were >3-fold higher (p<0.05) and each PG trended lower by >43% in Grade III disease. The other metabolites were at similar levels irrespective of grade (
Malignant tissue levels of the indicated metabolites were compared for poorer or better prognoses by mitosis, nuclear pleomorphism, and tubule formation indices (panel A) or nodal metastasis (panel B). 13-HODE (panel C) and PGE2 (panel D) levels were compared by poorer vs. better prognoses for: race, African (closed bars) or Caucasian American (open bars); Her2 score, 2 & 3 (closed bars) or 0 & 1 (open bars); age >50 years (closed bars) or ≤50 years (open bars); body mass index (BMI) >30 (closed bars) or ≤30 (open bars); estrogen receptors (ER) negative (closed bars) or positive (open bars); triple negative (tri (−)) for estrogen, progesterone, & Her2 receptors (closed bars) or not (open bars); tumor size, >2 (closed bars) or ≤ 2 cm (open bars). p Values are from Students t-test corrected for the 3 comparisons in each component of growth (panel A), for the 7 metabolite comparisons (panel B), or for the 7 marker comparisons (panels C and D) by the false discovery rate method. Analysis of these two metabolites for progesterone receptors or for 15-HETE, 12-HETE, 5-HETE, 5-oxo-ETE, and PGD2 in all 8 marker categories found no significant differences (data not shown).
The level of 13-HODE was 2-fold higher in the cancer tissue of patients with ≥1 lymph node positive for disease compared to those with node negative disease (
We examined 8 other prognostic markers: African or Caucasian American, Her2 receptor presence or absence; age >50 or ≤50 yrs; BMI >30 or ≤ 30; absence or presence of estrogen or progesterone receptors; triple negativity for Her2, estrogen, and progesterone receptors or presence of at least one of these receptors; and tumor size of >2 or ≤2 cm. The first category in each marker carries a poorer prognosis except age which at >50 years is associated with more frequent but not more severe disease. 13-HODE (
In normal breast tissue, 13-HODE levels did not correlate significantly with those of 15-HETE, 12-HETE, PGE2, or PGD2 (Pearson correlation coefficients of −0.15, 0.31, −0.20, and −0.10, respectively). In cancer tissue, however, 13-HODE was strongly and significantly correlated with 15-HETE (r = 0.63; P<0.01) but not with 12-HETE, PGD2, or PGE2 (r = 0.49, −0.01, and 0.15, respectively; P values for these correlations are corrected for the 4 observations made in each tissue). Similar result occurred in tissues from patients with >20 Mib1 scores: 13-HODE and 15-HETE levels were significantly correlated in malignant (r = 0.72, P<0.01) but not in normal (r = −0.04) tissue. In sharp contrast to this result, correlations between 13-HODE and 15-HETE in malignant (r = −0.25) and normal (r = 0.18) tissues of patients with ≤20 Mib1 scores were not statistically significant. 13-HODE also failed to correlate significantly with 12-HETE, PGE2, or PGD2 levels in patients with ≤20 Mib1 scores.
No FA parameter, measured as μg/mg of tissue or percentage of total recovered FA, in malignant (
Levels of the indicated FA are presented as mass (upper panels) or percentage of total recovered FA (lower panels) in malignant (left panels) and normal (right panels) breast tissue of patients with high or low Mib1 scores. Comparison of the 7 FA parameters on the basis of high or low Mib1 score by Students t-test gave no significant differences even before correction for multiple comparisons; the same analysis in RBC and plasma likewise revealed no significant differences as a function of Mib1 scores (results not shown).
There were no significant correlations between the levels of 13-HODE, PGE2, or PGD2 in malignant tissue and their FA precursors in cancer or normal breast tissue, RBC, or plasma (
FA mass | FA percentage | |||||
Tissue | 13-HODE | PGD2 | PGE2 | 13-HODE | PGD2 | PGE2 |
Malignant breast LA | 0.02 |
−0.10 | −0.01 | −0.20 | −0.22 | −0.06 |
Malignant breast AA | 0.06 | −0.17 | −0.17 | −0.04 | 0.11 | −0.07 |
Normal breast LA | −0.22 | −0.18 | −0.07 | −0.32 | −0.19 | −0.10 |
Normal breast AA | −0.23 | −0.12 | −0.16 | −0.22 | −0.04 | 0.05 |
RBC LA | −0.29 | −0.18 | −0.10 | −0.29 | −0.19 | −0.16 |
RBC AA | −0.19 | −0.13 | −0.10 | −0.07 | −0.05 | −0.17 |
Plasma LA | −0.03 | 0.04 | 0.19 | −0.09 | −0.02 | −0.22 |
Plasma AA | −0.17 | 0.00 | 0.02 | −0.11 | −0.10 | −0.24 |
Pearson correlation coefficients between the cited FAs and metabolites. None of the correlations attained statistical significance. There were also no significant correlations between the FA in patients with >20 Mib1 scores (data not shown).
Based on the results in
15-LO-1 catalyses the oxygenation of AA to 15-HETE and 12-HETE in a 89∶11 ratio, prefers LA over AA as substrate, and makes 13-HODE in excess when both FA are available. This FA preference along with the higher levels of LA compared to AA (
PGE2 and D2 trended lower in the cancer tissue of patients with Mib1>20 scores (
In conclusion, the metabolites and pathophysiology behind the contributions of FA oxygenases to poor survival in breast cancer has been ill-defined. We find that among the metabolites of the oxygenases known or found here to stimulate breast cancer cell proliferation, 13-HODE stands alone in associating with rapidly proliferating, rapidly dividing, aggressive grade, and perhaps metastasizing breast cancer. Three oxygenases make 13-HODE but correlation studies suggest that its major producer in rapidly proliferating breast cancer is 15-LO-1. Since 15-LO-1 makes other metabolites that are not characterized for proliferative activity in breast cancer cells or measured here, 13-HODE’s contribution to proliferation, division, and metastasis may be complemented or even superseded by other products of 15-LO-1. This caveat also applies to the trends of PGE2 and D2 to be negatively associated with these parameters of aggressive disease. Nonetheless, our results indicate that 13-HODE is a marker for breast cancer severity and the 15-LO-1/13-HODE pathway is associated with a rapidly proliferating, dividing, and possibly metastasizing phenotype. We propose that the over expression of this pathway speeds breast cancer’s growth and spread. Over expression of the other oxygenase-metabolite pathways, including the CO/PGE2/D2 pathways, do not use this specific mechanism to worsen the disease.
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